Screening of expression constructs suitable for protein pharmaceuticals is often done in batch cultivations. But the production of the recombinant protein is made during fed-batch cultivations. The two types of cultivations are different and therefore may good expression constructs that grow poorly in batch cultivations but good in fed-batch cultivations be rejected. Therefore would it be desirable to develop a fed-batch method that can be used in shake flasks. Biosilta has developed a method where starch is broken down into glucose by an enzyme creating fed-batch conditions. This method has been tried out and analyzed during this project. It is shown that the cells grown under these conditions can be glucose limited. However, at a later stage of the cultivation the cells produce a large amount of acetate and pH is not stable. The system builds on a booster tablet which content is unknown. If the booster is not added to the cultivations the cells stop growing, this indicates that there is some other limitation than just glucose. It is also seen that the amount of protein that is produced during this fed-batch mimic cultivation is much lower than that is produced during normal batch cultivations. I would therefore not recommend EnBase as a screening method. / Screening av nya rekombinanta proteiner som ska användas till läkemedel sker oftast i batch-odling. Men själva odlingen av proteinläkemedlet sker sedan under fed-batch förhållanden. Dessa två typer av odling är olika och då cellerna växer olika kan detta leda till att fel, eller den inte mest lämpade kandidaten väljs. Därför vore det önskvärt att ta fram en fed-batch liknande metod i skakkolvar. Biosilta har tagit fram en metod där ett enzym bryter ned stärkelse till glukos som påminner om fed-batch. Denna metod har testats och undersökts i detta examensarbete. Det har visat sig att cellerna som växer under dessa förhållanden är begränsade på glukos men producerar stora mängder ättiksyra under den senare fasen av odlingen och att pH varierar mycket. Systemet bygger mycket på att en booster-tablett tillsätts, vad denna tablett innehåller är okänt. Men om tabletten inte tillsätts slutar cellerna att växa, detta tyder på att det finns någon mer begränsning än glukos. Det visade sig även att protein produktionen blev mycket lägre än vid odling i batch-fas. Det skulle av anledning av ovanstående inte vara bra att använda sig av EnBase som en screening metod.

Filamentösa svampar används inom många biotekniska områden. Svamparnas biomassa kan användas till föda eller djurfoder, medan biprodukter som svampar producerar kan användas inom områden som industri samt medicin. Kultiveringen av Neurospora intermedia och Aspergillius oryzae genomförs på smör, raps och frityrolja i detta arbete för att få en större förståelse för hur svamparna kan växa på olika lipider samt skillnaden som kan uppstå mellan olika arter av filamentösa svampar. Under arbetes studeras biomassakoncentration, proteinhalt samt pH. Även problem som att svamparna kan lagra lipider i cellerna och att det kan påverka resultaten genom en ökning av biomassakoncentrationen diskuteras.  Kultiveringarna genomfördes i Erlenmeyerflaskor med 20 g/L lipid samt saltblandning för att bestå med nödvändiga näringsämnen. För A.oryzae genomförs även kultivering i en airliftbioreaktor för att förstå hur svampen beter sig i större skala. Högst koncentrationen torrbiomassa som erhålls ifrån Erlenmeyerflaskorna är 18,49±1,90 g/L för A.oryzae samt17,31±1,14 g/L för N.intermedia med en proteinhalt för torr biomassa upp till ca 14%.Utbytetmellan torr biomassa och lipid som tillsatts i kultiveringen hamnade på 94,00%±0,06%(rapsolja) för A.oryzae samt 81,69%±0,007% (frityrolja) för N.intermedia. De höga utbytena kan förklaras med att svamparna kan lagra lipider i cellerna vilket även ger de höga koncentrationen av torr biomassa. För att förstå morfologin, lipidhalt i biomassan samt pH beteende behövs mer arbete utföras med ändring av parametrar som pH justeringar, temperatur, luftflöde samt analys av biomassan för att utreda hur mycket lipider som lagras. / Filamentous fungi are used in many different biotechnological fields. The fungal biomass can be used for food or feed, while by-products that the fungi produce can be used in different industries or medical applications. The cultivations of Neurospora intermedia and Aspergillius oryzae implemented on butter, rapeseed and frying oil that is used in this work is for the larger understanding of how the fungi can grow on different lipids and the difference between species of fungi. Biomass concentration, protein content and pH are the main focus during this work, but problems like lipids storage in the biomass that can affect the results by increase of the total biomass concentration is discussed. The cultivations were implemented in Erlenmeyerflasks with 20 g/L lipid with added saltsolution with necessary nutrients. For A.oryzae cultivation in an airlift bioreactor was carried out for the understanding of larger scale cultivation. Highest concentration of dry biomass obtained from Erlenmeyerflasks are 18,49±1,90 g/L for A.oryzae and 17,31±1,14 g/L for N.intermedia with protein content for dry biomass up to 14%.The yield for dry biomass perlipid added during the cultivation ended at 94,00%±0,06% (rapeseed oil) for A.oryzae and 381,69%±0,007% (frying oil) for N.intermedia. During cultivation the cells can store lipids and that explains the high yields and biomass concentrations. For the understanding of morphology, the amount of lipids in the biomass and pH behaviour needs more work that includes changes of parameters like pH adjustment, temperature, airflow and analysis of the biomass for lipid content.

No description available.

Chromatography units, used in the production of pharmaceuticals, degrade with use and need to be changed or repackaged. This study investigates the effectiveness of two statistical methods, principal component analysis and simple and one-point multiparameter technique, for determining degradation in the Fibro chromatography unit. The methods have been shown to be effective on resin chromatography columns but not before tested on the relatively new Fibro chromatography unit. The statistical methods are implemented in an unreleased version of the monitoring and control software Unicorn. This implementation aims to be a proof of concept for including more complex methods for monitoring runs directly in the software, easing the workflow of operators by removing the need to export measurements to a third-party program. The methods were tested on measurements of absorbance, conductivity, and pressure from two series of chromatograms performed on two Fibro chromatography units. One of the units was defective and broke down halfway through the series. Principle component analysis could clearly visualize a difference between early and late runs on the defective unit. The same could only be achieved for the non-defective unit by excluding measurements of pressure. Simple and one-point multiparameter technique visualized trends from early to late in the series which were much clearer for the defective unit. Both methods showed signs of predicting degradation in a Fibro chromatograpy unit but require validation on chromatogram series with more direct measurements of performance and a wider range of failure causes.

This thesis demonstrates a parameter estimation technique for bioprocesses that utilizes measurement noise in experimental data to determine credible intervals on parameter estimates, with this information of potential use in prediction, robust control, and optimization. To determine these estimates, the work implements Bayesian inference using nested sampling, presenting an approach to develop neural network (NN) based surrogate models. To address challenges associated with non-uniform sampling of experimental measurements, an NN structure is proposed. The resultant surrogate model is utilized within a Nested Sampling Algorithm that samples possible parameter values from the parameter space and uses the NN to calculate model output for use in the likelihood function based on the joint probability distribution of the noise of output variables. This method is illustrated against simulated data, then with experimental data from a Sartorius fed-batch bioprocess. Results demonstrate the feasibility of the proposed technique to enable rapid parameter estimation for bioprocesses. / Thesis / Master of Applied Science (MASc) / Bioprocesses require models that can be developed quickly for rapid production of desired pharmaceuticals. Parameter estimation is necessary for these models, especially first principles models. Generating parameter estimates with confidence intervals is important for model based control. Challenges with parameter estimation that must be addressed are the presence of non-uniform sampling and measurement noise in experimental data. This thesis demonstrates a method of parameter estimation that generates parameter estimates with credible intervals by incorporating measurement noise in experimental data, while also employing a dynamic neural network surrogate model that can process non-uniformly sampled data. The proposed technique implements Bayesian inference using nested sampling and was tested against both simulated and real experimental fed-batch data.

Utilizou-se um meio de cultivo a base de farinha de mandioca, suplementado com nutrientes, em fermentador agitado (700 rpm) e aerado (10 litros de ar/min), com volume de reação de 10 litros praticamente constante, fração de inóculo de 10% em volume, ph 4,0 e temperatura de 35ºC. Foram realizados ensaios com concentração total de açúcares de 20 g/l e 40 g/l, tanto descontínuos como descontínuos alimentados. Nestes variou-se a vazão mássica de alimentação (fs), o instante de início de alimentação e a condição do xarope de farinha (previamente hidrolisado ou não). Repetições dos ensaios descontínuos indicaram variabilidade de resultados elevada. Não se observou expressivas mudanças no crescimento microbiano, a não ser pelo aumento na velocidade específica nos ensaios descontínuos alimentados a 20 g/l. A síntese de glicoamilase foi sensivelmente aumentada nos ensaios descontínuos alimentados a 20 g/l (produtividade dobrada). A 40 g/l, obteve-se produtividade 26% superior. Os melhores resultados foram obtidos com fs=17,1 gart/h a so=20 g/l e fs=32,2 gart/h a 40 g/l, e obteve-se o pior no ensaio em que se alimentou desde o início de cultivo. A so=20 g/l a repressão se apresenta como principal mecanismo de controle de síntese de glicoamilase, não ocorrendo a mesma a 40 g/l, ensaios nos quais a indução tornou-se muito relevante. / In order to study different processes and the influence of control mechanism on glucoamylase synthesis, several batch and fed-batch runs were made with Aspergillus awamori NRRL 3112. A medium containing cassava flour and nutrients were used in a 10 liters stirred and aerated tank, at pH 4,0 and temperature 35 °C. The batch and fed-batch runs used 20 and 40 g of total reducing sugars (TRS) per liter. In the fed-batch runs, the carbon source feed rate (fs), the feeding start time, and whether the syrup were pre-hydrolyzed or not were varied. Repeated batch runs showed significant variability. Notable changes in cell growth were not observed, unless by the increase of the specific growth rate in the 20 gTRS/l fed-batch runs. The enzyme productivity doubled in the lower sugar concentration fed-batch runs, but increased just 26% in the runs with 40g/l of TRS. The best results were achieved at 20g/l with carbon source feed rate=17,1 gTRS/h and fs=32,2 gTRS/h at 40g/l. The worst noted when the feeding started at the beginning of the run. At 20 gTRS/l, repression showed as the main mechanism control in order to synthesize glucoamylase. On the other hand induction became the relevant factor when 40gTRS/l were offered to microorganism.

Utilizou-se um meio de cultivo a base de farinha de mandioca, suplementado com nutrientes, em fermentador agitado (700 rpm) e aerado (10 litros de ar/min), com volume de reação de 10 litros praticamente constante, fração de inóculo de 10% em volume, ph 4,0 e temperatura de 35ºC. Foram realizados ensaios com concentração total de açúcares de 20 g/l e 40 g/l, tanto descontínuos como descontínuos alimentados. Nestes variou-se a vazão mássica de alimentação (fs), o instante de início de alimentação e a condição do xarope de farinha (previamente hidrolisado ou não). Repetições dos ensaios descontínuos indicaram variabilidade de resultados elevada. Não se observou expressivas mudanças no crescimento microbiano, a não ser pelo aumento na velocidade específica nos ensaios descontínuos alimentados a 20 g/l. A síntese de glicoamilase foi sensivelmente aumentada nos ensaios descontínuos alimentados a 20 g/l (produtividade dobrada). A 40 g/l, obteve-se produtividade 26% superior. Os melhores resultados foram obtidos com fs=17,1 gart/h a so=20 g/l e fs=32,2 gart/h a 40 g/l, e obteve-se o pior no ensaio em que se alimentou desde o início de cultivo. A so=20 g/l a repressão se apresenta como principal mecanismo de controle de síntese de glicoamilase, não ocorrendo a mesma a 40 g/l, ensaios nos quais a indução tornou-se muito relevante. / In order to study different processes and the influence of control mechanism on glucoamylase synthesis, several batch and fed-batch runs were made with Aspergillus awamori NRRL 3112. A medium containing cassava flour and nutrients were used in a 10 liters stirred and aerated tank, at pH 4,0 and temperature 35 °C. The batch and fed-batch runs used 20 and 40 g of total reducing sugars (TRS) per liter. In the fed-batch runs, the carbon source feed rate (fs), the feeding start time, and whether the syrup were pre-hydrolyzed or not were varied. Repeated batch runs showed significant variability. Notable changes in cell growth were not observed, unless by the increase of the specific growth rate in the 20 gTRS/l fed-batch runs. The enzyme productivity doubled in the lower sugar concentration fed-batch runs, but increased just 26% in the runs with 40g/l of TRS. The best results were achieved at 20g/l with carbon source feed rate=17,1 gTRS/h and fs=32,2 gTRS/h at 40g/l. The worst noted when the feeding started at the beginning of the run. At 20 gTRS/l, repression showed as the main mechanism control in order to synthesize glucoamylase. On the other hand induction became the relevant factor when 40gTRS/l were offered to microorganism.

No description available.

To diagnose patients suffering from blood clotting disorders latex immununoassays (LIA) can be used. A time consuming manual tangential flow filtration (TFF) process suggests the implementation of an automatic TFF system to improve the efficiency, profitability, and expandability of the production facility of LIA at Nordic Biomarker. Tests were made of the automatic TFF system's ability to perform the desired steps of concentration, dilution and diafiltration, both with purified water and mimicked product. The mimicked product of micro particles (MP) mixed with monoclonal antibodies (mAb) was also used to further test the system's pressure control, safety alarms and stops, and to determine a permeate flux by a critical flux experiment. The results imply a functional TFF system able to automatically concentrate the process fluid and maintain a stable volume during diafiltration, although an additional permeate pump was ordered to be able to attain a fully functional performance of the automatic TFF process. The final part of the implementation was to initiate a validation draft including a risk assessment, OQ plan and PQ plan that resulted in a plan of the main tests to be performed. To conclude, the essential part of the implementation of a high quality and efficient automatic TFF process was conducted to facilitate future expansion of the production of LIA.

Thesis (PhD (Process Engineering))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Biological agents offer several opportunities to address the many challenges faced in intensive reticulated aquaculture. We therefore isolated and selected Bacillus spp. as potential biological agents, because this group has demonstrated an array of biological activities of possible benefit to aquaculture. They also display advantages in terms of robustness during bioprocessing and end product application. Natural isolates obtained from Cyprinus carpio, selected as a model high-value ornamental fish species, and associated culture environments, were purified and assessed for in vitro efficacy based on the inhibition of growth of pathogenic Aeromonas hydrophila and the decrease in concentrations of ammonium, nitrite, nitrate and phosphate ions, typically found as waste products in aquaculture systems. Based on suitability for aquaculture application, isolates B001, B002 and B003 were selected and further evaluated in vitro and in an in vivo trial with C. carpio. Inhibition of Aer. hydrophila growth and a decrease in concentrations of waste ions were demonstrated in these studies. Based on 16S RNA sequence homology, the isolates were identified as Bacillus subtilis, B. cereus and B. licheniformis, respectively. High sequence homology between B. subtilis and B. anthracis necessitated further safety studies on the best isolate, B. cereus NRRL100132 (B002). The isolate was shown not to contain the anthrax virulence genes pOX1, pOX2 or the B. cereus enterotoxin. Elucidation of the potential modes of action of a biological agent facilitates an understanding of functionality and encourages technology uptake by end users. Competitive exclusion through growth rate and competitive uptake of glucose and iron, the latter facilitated by siderophore production, were shown to be key mechanisms at play in inhibition of Aer. hydrophila by the B. cereus isolate. As production cost is an important consideration in development of commercially relevant biological products, we examined the optimization of nutrient supplementation, which has an impact on high-density production of spores by fermentation. Corn steep liquor (CSL) was identified as a lower cost and more effective nutrient source in comparison to conventional nutrient substrates, in particular yeast extract and nutrient broth. The improved sporulation performance of B. cereus could be related to the increased availability of free amino acids, carbohydrates, and minerals in CSL, which had a positive effect on organism growth and sporulation efficiency. The impact of nutrient concentration on spore yield and productivity was modelled to develop a tool for selection of optimal conditions. Excellent correlation with actual laboratory fermentation data was demonstrated. A cost analysis revealed that production using liquid phytase treated and ultra-filtered CSL was less expensive than spray dried CSL and supported cultivation of B. cereus spores at densities higher than 1×1010 CFU ml 1. Adoption of biological agents in commercial applications is lacking, due to limitations in process and product development that address key end user product requirements such as cost, efficacy, shelf life and convenience. The development of suitable spore recovery, drying, formulation and tablet production process steps was thus performed. Key criteria used for downstream process unit evaluation included spore viability, recovery, spore balance closure, spore re-germination, product intermediate stability, end product stability and efficacy. A process flow sheet comprising vertical tube centrifugation, fluidised bed agglomeration and tablet pressing yielded an attractive product. The formulation included corn steep liquor and glucose to enhance subsequent spore re-germination. Viable spore recovery and spore balance closure across each of the process units was high (>70% and >99% respectively), with improvement in recovery possible by adoption of continuous processing at large scale. Spore re-germination was 97%, whilst a product half-life in excess of 5 years was estimated based on thermal resistance curves. The process resulted in a commercially attractive product and affordable variable cost of production. Functionality of the product, incorporating the B. cereus isolate, was investigated across a range of physiological conditions, including salinity, pH and temperature, based on rearing of C. carpio. Temperature had a significant influence on germination, specific growth rate and increase in cell number of B. cereus, whilst salinity and pH did not have any measurable effect on growth. Controlled studies in bioreactors and modelling of the data to the Arrhenius function indicated the existence of high and low growth temperature domains. The rates of pathogenic Aer. hydrophila suppression and decrease in waste ion concentrations (ammonium, nitrite, nitrate and phosphate) were translated into a linear predictive indicator of efficacy of the B. cereus isolate at different temperatures. This study has resulted in development of an upstream and downstream process for production of a new B. cereus isolate (NRRL 100132) which was shown to be safe, stable, functional, robust and cost effective for application in aquaculture. / AFRIKAANSE OPSOMMING: Biologiese middels bied verskeie maniere om die veelvoudige uitdagings van intensiewe netsgewyse akwakultuur aan te spreek. Gevolglik het ons uitgesoekte Bacillus spesies as potensiële biologiese middels geïsoleer, omdat hierdie groep verskeie biologiese aktiwiteite demonstreer wat van potensiële waarde kan wees in akwakultuur. Die groep toon ook voordele in terme van robuustheid gedurende bioprosessering en eind-toepassings. Natuurlike bakteriële isolate vanuit Cyprinus carpio geassosieerde kultuur omgewings, geselekteer as 'n hoë-waarde model ornamentele spesie, is gesuiwer. Die in vitro doeltreffendheid van die isolate is bepaal gebasseerd op die groei inhibisie van patogeniese Aeromons hydrophila asook die afname in konsentrasies van ammonium, nitriete, nitrate en fosfaat ione wat as tipiese afval produkte gevind word in akwakultuur sisteme. Isolate B001, B002 en B003 is geselekteer op grond van geskiktheid en verder evalueer in in vitro en in vivo proewe met C. carpio. Groei inhibisie van Aer. hydrophila asook 'n afname in konsentrasies van afval ione was tydens die studies gedemonstreer is. Die isolate is identifiseer as Bacillus subtilis, B. cereus en B. licheniformis, respektiewelik, op grond van 16S RNS volgorde homologie. Die hoë volgorde homologie tussen B. subtilis en B. anthracis het verdere veiligheidstudies op die beste isolaat, B. cereus NRRL100132 (B002) genoodsaak. Die isolaat het nie die antraks virulensie plasmied pOX1, pOX2 of die B. cereus enterotoksien getoon nie. Uitklaring van die potensiële meganismes van aksie van biologiese middels fasiliteer 'n begrip van funksionaliteit en moedig tegnologie aanvaarding deur eind-gebruikers aan. Mededingende uitsluiting deur groeitempo en mededingende opname van glukose asook die produksie van siderofore is bewys as sleutel meganismes betrokke in die inhibisie van Aer. hydrophila deur die B. cereus isolaat. Aangesien koste 'n belangrike oorweging is in die ontwikkeling van kommersiële toepaslike biologiese produkte, is die optimisering van voedingstof aanvullings wat 'n impak het op hoëdigtheid produksie van spore deur fermentasie ondersoek. Week-vloeistof van mielie prosessering (CSL) is identifiseer as 'n lae koste en effektiewe voedingsbron in vergelyking met konvensionele voeding substrate, veral gisekstrak en voedingsboeljon. Die verbeterde sporulering prestasie van B. cereus kon toegeskryf word aan die verhoogde beskikbaarheid van vrye aminosure, koolhidrate en minerale in CSL, wat 'n postitewe effek op organisme groei en sporulerings effektiwiteit getoon het. Die impak van voedingstof konsentrasie op spoor opbrengs en produktiwiteit is gemodelleer om 'n werktuig vir die selektering van optimale kondisies te ontwikkel. Uitstekende korrelasie met werklike laboratorium data is gedemonstreer. Koste analises het getoon dat produksie deur middel van vloeibare fitase-behandelde en ultra-filtreerde CSL goedkoper is as sproei-gedroogde CSL en ondersteun verder die kultivering van B. cereus spore teen digthede hoër as 1 x 1010 kolonie vormende eenhede.ml-1. Die opname van biologiese middels in kommersiële toepassings skiet tekort as gevolg van beperkinge in proses en produk ontwikkeling wat belangrike eind-gebruiker vereistes soos koste, doeltreffendheid, rak leeftyd en gerieflikheid aanspreek. Die ontwikkeling van toepaslike prosesse vir spoor herwinning, droging, formulering en tablet produksie is gevolglik uitgevoer. Belangrike maatstawwe wat gebruik is vir stroomaf proseseenheid-ontwikkeling het lewensvatbaarheid, herwinning, spoor balans sluiting, spoor her-ontkieming, intermediêre produk stabiliteit, eindproduk stabiliteit en doeltreffendheid ingesluit. 'n Proses vloeidiagram bestaande uit vertikale buis sentrifugasie, vloeibare bed agglomerasie en tablet persing het 'n aantreklike produk voortgebring. Die formulering het ook CSL en glukose ingesluit om gevolglike spoor herontkieming te verbeter. Lewensvatbare spoor herwinning en spoor balans sluiting oor elke proses eenheid was hoog (>70% en 99% respektiewelik) met verbetering in herwinning wat moontlik gemaak is deur die gebruik van aaneenlopende prosessering op groot skaal. Spoor her-ontkieming was 97%, terwyl produk halfleeftyd langer as 5 jaar beraam is, gebasseer op termiese weerstand grafieke. Die proses het gelei tot 'n kommersiële aantreklike produk asook bekostigbare veranderbare produksie koste. Die funksionaliteit van die tablet-produk met die ingeslote B. cereus isolaat is ondersoek oor 'n reeks fisiologiese kondisies insluitend soutgehalte, pH en temperatuur, gebasseer op die kultivering van C. carpio. Temperatuur het 'n betreklike invloed op ontkieming, spesifieke groeitempo en toename in sel hoeveelheid van B. cereus gehad, terwyl soutgehalte en pH nie enige meetbare effek op groei gehad het nie. Gekontrolleerde studies in bioreaktors en modellering van die data op die Arrhenius funksie het hoë en lae groei temperatuur domeins gewys. Die tempo van patogeniese Aer. hydrophila onderdrukking en afname in konsentrasies van afval-ione (ammonium, nitriete, nitrate en fosfaat) is herlei na 'n liniêre voorspellende aanwysing van effektiwiteit van B. cereus isolate by verskillende temperature. Die studie het gelei tot die ontwikkeling van stroomop- en stroomaf-prosesse vir die produksie van 'n nuwe B. cereus isolaat (NRRL 100132) wat bewys is as veilig, stabiel, funksioneel, robuust en koste effektiewe vir toepassing in akwakultuur.